1. Field of the Invention
The present invention relates to a method of manufacturing a reference material for determining incorporation of a genetically modified (GM) plant into a sample or analyzing a mixing ratio from a tissue-cultured cell line that is obtained by incubating tissues of either a GM plant or a non-GM plant, and a method of determining incorporation of a GM plant into a sample and analyzing a mixing ratio using the reference material.
2. Discussion of Related Art
A genetically modified organism (GMO) refers to an organism whose productivity or functionality is improved by artificially separating genes of interest from animals, plants or microorganisms and binding the separated genes, unlike the development of a breed using a conventional plant breeding technique. In general, genes associated with herbicide resistance, insect tolerance, disease tolerance, cold resistance and/or nutrition supplement are introduced into a plant so as to improve the productivity of GMOs and enhance the quality of the GMOs.
Examples of GMOs which are the most commercially available all over the world include maize and beans. In addition, this genetic engineering technique may apply to other crops such as cotton, canola, potatoes and tomatoes. However, since the safety of GMOs is not scientifically verified, a labeling system in which a plant is labeled as a GMO when at least 3% of the GMO is mixed with its original plant is currently being enforced in Korea. Although there is a difference in threshold in other countries, similar labeling systems are in effect.
Therefore, a technique of quantitatively determining a mixing ratio is required in addition to a qualitative analysis method of detecting an incorporated GMO.
The quantitative analysis of GMOs is mainly divided into two categories: a protein analysis method and a DNA analysis method. In general, an ELISA method using a protein expressed from a gene introduced into a GMO has problems in that a detection rate is inferior to that of a PCR method, and proteins may be denatured due to heat treatment. Therefore, a technique for quantifying a gene that is introduced into a GMO using a real-time PCR method has been widely used. In order to quantify a mixing ratio in a GMO sample using the real-time PCR method, a reference material which can compare the copy number of a gene introduced into the GM plant is required.
As the GMO labeling system has come into effect, the accuracy and precision of analysis of the incorporation threshold at which a GMO is incorporated into a sample are very important, and a reference material used for the analysis is the most important so as to analyze the incorporation threshold with accuracy and precision. The reference material has to have secured long-term storage opportunities and show its own stability and homogeneity as well.
In recent years, a reference material prepared by mixing ground seed powder of a GMO and a non-GMO derived from respective crops at a certain weight ratio based on a conventional method, or a standard plasmid prepared by inserting an amplicon containing an endogenous gene and an inserted gene, which is extracted from a sample in which the GMO and the non-GMO are mixed at a certain weight ratio, has been used as the reference material. However, the reliability of these reference materials remains to be improved. In particular, lack of information on the purity of a starting sample and technology for proving the information is blocking the manufacture of the reference material. Therefore, a more stably and uniformly available reference material is required.